Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

Accumulation of misfolded α-synuclein (α-syn) is a hallmark of Parkinson’s disease (PD) thought to play important roles in the pathophysiology of the disease. Dendritic systems, able to modulate the folding of proteins, have emerged as promising new therapeutic strategies for PD treatment. Dendrimers have been shown to be effective at inhibiting α-syn aggregation in cell-free systems and in cell lines. Here, we set out to investigate the effects of dendrimers on endogenous α-syn accumulation in disease-relevant cell types from PD patients. For this purpose, we chose cationic carbosilane dendrimers of bow-tie topology based on their performance at inhibiting α-syn aggregation in vitro. Dopamine neurons were differentiated from induced pluripotent stem cell (iPSC) lines generated from PD patients carrying the LRRK2G2019S mutation, which reportedly display abnormal accumulation of α-syn, and from healthy individuals as controls. Treatment of PD dopamine neurons with non-cytotoxic concentrations of dendrimers was effective at preventing abnormal accumulation and aggregation of α-syn. Our results in a genuinely human experimental model of PD highlight the therapeutic potential of dendritic systems and open the way to developing safe and efficient therapies for delaying or even halting PD progression.


■ INTRODUCTION
Alpha-synuclein (α-syn) is a small presynaptic protein widely and abundantly expressed in neurons throughout the nervous system. 1 Even though the normal functions of α-syn are not completely understood, abnormal accumulation in neurons has been found in a variety of neurological diseases collectively known as synucleinopathies, 2,3 including Parkinson's disease (PD) in both sporadic and familial cases. 4 PD is an age-related, chronic, and progressive neurodegenerative disorder, characterized by loss of dopamine neurons (DAns) within the substantia nigra pars compacta, which contributes to the cardinal motor symptoms of the disease. 5,6 A pathological hallmark of PD is the presence of α-syn-containing intraneuronal inclusions, known as Lewy bodies. 2 Both the presence and intensity of Lewy bodies closely correlate with disease progression 7 and are thus thought to play a pivotal role in PD pathophysiology. 8 The facts that mutations or multiplications in the SNCA gene (encoding α-syn) cause familial PD 9 and that experimental models of α-syn overexpression 10,11 or artificially high loads of aggregated or preformed α-syn fibrils 12−14 lead to synaptic dysfunction and neuron death indicate that increased α-syn expression, accumulation, or aggregation in DAns may be sufficient to promote nigral degeneration.
Despite intensive research, PD remains an incurable neurological condition. Currently available treatments are designed to compensate the loss of dopamine and dopaminergic function, but they are palliative and limited by side effects and lack of long-term efficacy. 15,16 Unfortunately, current therapeutic strategies do not cure or block PD progression, emphasizing the critical need for finding new disease-modifying approaches that halt, slow, or delay disease progression. Considering its importance in PD pathophysiol-ogy, α-syn could be an interesting therapeutic target for this purpose, and inhibition of α-syn aggregation using proteinsolubilizing drugs is a potential therapeutic strategy.
Nanotechnology has contributed to our understanding of PD pathogenesis by providing tools that promote the solubilization of α-syn fibers and prevent the formation of αsyn aggregates. 17 Different nanomaterials have been explored for their usefulness as antiparkinsonian, antioxidant, and/or neuroprotective agents, including nanoparticles, 18 dendrimers, 19 and carbon nanotubes. 20 Among them, dendrimers are especially interesting as they are highly branched polymers with well-defined geometry and unique properties that can be functionalized according to the specific application for which they are designed. A variety of dendritic systems of different natures and topologies have been tested in the context of neurodegenerative diseases, including PAMAM, 21 polypropyleneimine dendrimers, 22 glycodendrimers, 23 and phosphorous 24 or carbosilane 25 dendrimers, and shown to have an effect on α-syn aggregation. 21,24,26,27 Moreover, dendrimers have also been reported to inhibit the aggregation of insoluble forms of amyloid 28 and prion 29 peptides and to facilitate their degradation. 30,31 For the present studies, we focused on carbosilane dendrimers as they have been proven to be effective in a variety of biomedical applications in a topology-dependent manner 25,32−36 and non-toxic at concentrations below 5 μM when used as transfecting or anti-cancer agents. 37,38 One of the main properties of carbosilane dendrimers is their inherent hydrophobicity, provided by their characteristic skeleton, which allows interaction with biological membranes in an efficient manner 39 and the presence of a permanent number of charges. For this reason, low generations of carbosilane dendrimers are effective for most applications, unlike the case of other dendritic skeletons. Specifically, in the context of protein misfolding diseases, cationic carbosilane systems have shown their ability to inhibit rotenone-induced α-syn fibrillation in mouse hippocampal cells 40 and amyloidogenic islet amyloid polypeptide (IAPP) aggregation in a mouse model of type II diabetes. 25 In this work, we investigated how the topology of cationic carbosilane dendrimers (spherical, dendritic wedge, or bow-tie) could influence the interaction with α-syn to avoid aggregation. After biophysical characterization of the interaction between α-syn and the different dendritic systems, we selected the bow-tie topology for testing effectiveness in an induced pluripotent stem cell (iPSC)-based experimental model of PD. Our results show that noncytotoxic concentrations of cationic carbosilane dendrimers are effective in preventing abnormal accumulation and aggregation of α-syn in dopamine neurons from PD patients.
Methods. Zeta Potential. The measurements of the zeta potential (electrokinetic potential) were performed with a Zetasizer Nano ZS from Malvern, which uses electrophoresis and LDV (laser doppler velocimetry) techniques to allow measurement of the electrophoretic mobility of the molecules in the solution. The zeta potential value was calculated directly from the Helmholtz−Smoluchowski equation using Malvern software. 42 The measurements were performed at 37°C with three repetitions. Increasing concentrations of the bow-tie dendrimer in a range of 0.25−25 μM were added to a 1 μM solution of α-syn, and the zeta potential was measured.
The analysis of possible changes in the zeta potential as a function of the molar ratio can be approximated by the number of dendrimer molecules that can attach to one molecule of α-synuclein. The number of binding centers is the point (X, O) corresponding to the point of intersection of two straight lines tangent to the curve of the zeta potential as a function of the molar ratio.
CD Spectrometry. The circular dichroism (CD) spectra (190−260 nm) were recorded for α-syn in the presence/absence of dendritic systems on a Jasco J-815 CD spectropolarimeter, in 5 mm-path length quartz cuvettes, with a wavelength step of 0.5 nm, a response time of 4 s, and a scan rate of 50 nm/min, maintained at 37°C. Each spectrum was the average of three repetitions. α-Syn was used at a concentration of 1 μmol/L. The changes in the secondary structure of α-syn in the presence and absence of dendritic systems I−III were tested immediately (dendrimers/dendron concentrations: 0.5−5 μmol/L) and after 48 and 72 h (dendrimers/dendron concentrations: 2 and 4 μmol/L) of incubation at 37°C. Dendrimers/dendron alone at the experiments' concentrations did not produce any discernible features in their CD spectra and were used as blanks for α-syn− dendrimer complex spectra.
iPSC-Derived DAn Generation. The iPSC lines used in our study were previously generated and fully characterized. 43 Specifically, we used iPSC generated from two patients carrying the G2019S mutation in the LRRK2 gene (LRRK2-PD) and from two healthy age-matched controls (CTRL) ( Table 1). The competent Spanish authorities (Commission on Guarantees concerning the Donation and Use of Human Tissues and Cells of the Carlos III National Institute of Health) approved the use of human iPSCs in this work. For DAn differentiation, iPSCs were transduced with LV.NES.LMX1A.GFP and processed as previously described. 44 Embryoid bodies (EBs) generated from iPSC colonies were cultured in nonadherent dishes for 3 days in the presence of mTeSR medium (Stem Cell Technologies) (stage 1). At stage 2, EBs were cultured for 10 days in suspension in N2B27 medium [DMEM/F12 medium (Gibco), neurobasal medium (Gibco), 1% B27 (Gibco), 0.5% N2 (Gibco), 1% GlutaMAX (Gibco), and 1% penicillin/streptomycin] supplemented with 10 ng/mL FGF2 (PeproTech), 100 ng/mL FGF8 (PeproTech), and 100 ng/mL SHH (PeproTech). For DAn maturation, at stage 3, the neural precursor cells (NPCs) were co-cultured with PA6 stromal cells for 3 weeks in N2B27 medium supplemented with FGF8 and SHH. Finally, at stage 4, dopaminergic neurons generated were dissociated using Accutase (Merck), re-plated on Matrigel-coated dishes, and cultured for 1 week in N2B27 with FGF8 and SHH. Correct differentiation was judged by co-immunostaining with neuron-specific class III-β-tubulin (TUJ1), a widely used neuronal marker, and tyrosine hydroxylase (TH), the main neuronal marker for dopaminergic neurons.  Immunofluorescence. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4°C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences). Samples were then incubated with secondary antibodies for 2 h at 37°C: Alexa Fluor 647 anti-mouse IgG2a (1:100, A21241, Invitrogen), Cy3 antirabbit IgG (1:200, 711-165-152, Jackson), and Alexa Fluor 488 antimouse IgG1 (1:100, A21121, Invitrogen). To visualize nuclei, slides were stained with DAPI (1:5000, Invitrogen) and then mounted with PVA:DABCO. Images were taken using a Leica SP5 confocal microscope and analyzed with FIJI Is Just ImageJ. For quantification of cytoplasmic accumulation of α-syn, the fluorescence intensity in the green channel was corrected using the number of DAns (area green channel/area red channel). The scoring of α-syn-positive DAns was performed by researchers blinded to the experimental conditions.
Statistical Analysis. Results are expressed as mean ± SEM. The normal distribution of data was tested using D'Agostino and Pearson omnibus normality test. Student's t-test or one-way ANOVA followed by Tukey's post hoc test was used for statistical analysis in normal distributions. Statistical analysis for the non-normal distribution data was performed using the Mann−Whitney U test or Kruskal−Wallis test. The software used was GraphPad Prism 6, and the value of p < 0.05 was considered statistically significant. The number of experiments (n) is indicated in the legends to the figures.

■ RESULTS AND DISCUSSION
Cationic Carbosilane Dendritic Systems Used in the Studies. Previous work in some of our laboratories helped us to identify a strong influence of dendrimer topology on their capacity to interact with amyloid peptides and therefore to inhibit beta-amyloid aggregation in an animal model of type II diabetes. 25 The morphology (spherical, dendron, or bow-tie) can arrange the same number of peripheral charges differently and therefore modify the interaction with the peptidic domain responsible of the misfolding effect. For this reason, in the current studies, we first set out to investigate the influence of dendritic system topology on α-syn folding. For this purpose, we chose three cationic carbosilane dendritic systems with the same number of charges but different topologies: G 1 Si-(SNMe 3 I) 8 (I) as a spherical dendrimer, 41 HOC 6 H 4 G 3 Gn-(SNMe 3 I) 8 (II) as a dendron, 41 and (IMe 3 NS) 4 4 (III) as a bow-tie system 25 (Figure 1).
Study of Interaction Between α-Syn and the Dendritic System: Biophysical Characterisation. The interaction of carbosilane dendrimers and α-syn and their effect on the aggregation status of synthetic peptides of α-syn have been thoroughly analyzed by different biophysical techniques such as zeta potential and circular dichroism. 40 To ascertain the influence of the system's topology on such interactions, we first measured changes in zeta potential (directly related to the particle charge environment) to study the interaction between α-syn and different topologies of carbosilane dendrimers. α-Syn is a small acidic protein composed of three well-differentiated domains: (i) a positively charged region called N-terminal lipid-binding α-helix, (ii) a   45 Due to the predominance of negative charges at pH = 7.4, the α-syn zeta potential value is around −21 mV. However, upon addition of different concentrations of dendritic systems I−III, the zeta potential turned positive until reaching a value of ∼20 mV (Figure 2A). Due to the cationic nature of dendritic structures, it is likely that nanoconjugates formed with α-syn have positive charge values independent of the system topology. These results indicate that the topology of the dendrimer system does not significantly influence the zeta potential of the resulting nanoconjugate with α-syn. Moreover, our results also show that nanoconjugates were formed by one molecule of dendrimer/dendron per one α-syn molecule (1:1 molar ratio), in all tested topologies (Figure 2). We next used CD to analyze conformational changes in the secondary structure of α-syn upon interaction with dendrimers I−III under nearly physiological conditions (in solution). The    of dendrimers I−III over a concentration range spanning one order of magnitude (0.5−5 μM) did not cause significant changes in CD spectra except for the largest concentration tested (Figure 3). Taking these results into account, we then  (Figure 4). The capacity of dendrimers I−III to inhibit this conversion was tested after 48−72 h of incubation in the presence of two different concentrations of dendrimers (α-syn/dendrimer molar ratios of 1:2 or 1:4). All the tested dendrimer topologies exhibited an overall inhibitory activity in the conversion of disordered α-syn into β structure ( Figure 4). However, dendron II elicited marked changes in α-syn CD spectra, which were more evident at the highest concentration tested, and thus was excluded from further studies. Between the spherical (I) and bow-tie (III) systems, we chose the latter for downstream experiments in cells due to its relative stronger and more durable inhibiting activity at low concentration ( Figure 4) and also because previous tests of the spherical dendrimer I in a mouse model of type II diabetes failed to show anti-aggregation activity of the β-amyloid protein. 25 Effect of Cationic Carbosilane Dendrimers on the Survival of PD Patient Dopamine Neurons in Culture. Even though the interaction of carbosilane dendrimers and αsyn is well-documented, 40 as is their capacity to inhibit the aggregation of synthetic peptides of α-syn, to our knowledge, no studies have investigated the effect of carbosilane dendrimers on the endogenous α-syn present in diseaserelevant cell types. The relevance of utilizing cell types as similar as possible to those affected by specific diseases when studying pathogenic mechanisms or testing potential therapies is widely recognized. 46,47 In this respect, reprogramming patients' somatic cells to pluripotency for the generation of patient-or disease-specific iPSC has emerged as a powerful strategy to produce theoretically unlimited amounts of diseaserelevant cell types. 48 In the case of PD, several laboratories have generated patient-specific iPSC lines from idiopathic and genetic forms of the disease and used them to differentiate DAns that recapitulated key hallmarks of the disease (reviewed in 49−51 ). In particular, we have previously shown that DAns differentiated from iPSC obtained from PD patients carrying the G2019S mutation in the LRRK2 gene (LRRK2-PD) exhibit abnormal accumulation of α-syn 43 as a consequence of impaired chaperone-mediated autophagy (CMA). 52 We have also shown that while other LRRK2-PD iPSC-derived brain cells such as astrocytes abnormally accumulate α-syn as well, it is accumulation in DAns that ultimately leads to neurodegeneration. 53 For these reasons, in the current studies, we set out to test whether cationic carbosilane dendrimers had any effect on the abnormal, disease-related, accumulation of α-syn in LRRK2-PD iPSC-derived DAns. For this purpose, we used iPSC lines representing two LRRK2-PD patients and two healthy individuals as controls, generated and fully characterized in previous work, 43 and differentiated them into DAns following a robust directed protocol 44 (see Figure 5A and Experimental section for details). Cationic carbosilane dendrimers (bow-tie system, III) were added to DAn cultures at two different concentrations (1 and 3 μM) and incubated for 48 h to first ascertain any effects on DAn survival. Under these conditions, the highest concentration of dendrimer resulted in ∼60% decrease in DAn numbers, as measured by immunostaining for the DAn marker tyrosine hydroxylase (TH), when compared with parallel cultures treated with the vehicle alone ( Figure 5B). In contrast, dendrimers were not toxic to DAns when used at 1 μM for 48 h, at least in terms of cell viability ( Figure 5B). Indeed, we even observed a slight increase (albeit not statistically significant) in DAn numbers in LRRK2-PD-DAns treated with 1 μM III compared to untreated LRRK2-PD-DAns, which could be explained by the dendrimers' inhibitory effects on DAn α-syn accumulation (see below). Previous work in the context of Alzheimer's disease showed that several dendrimers were efficient in preventing Aβ-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. 31,54 Our results so far demonstrate that human DAns derived from PD patient-specific iPSC can safely be cultured, for at least 48 h, in the presence of 1 μM cationic carbosilane dendrimers of bow-tie topology (III) and suggest that this treatment could be beneficial for their survival, even though culture times longer than 48 h might be necessary to unambiguously demonstrate the latter.
Effect of Cationic Carbosilane Dendrimers on the Abnormal Accumulation of α-Syn in DAns from PD Patients. Consistent with previous reports from our laboratories and others, 43,52,55,56 DAns differentiated from LRRK2-PD iPSCs showed evident cytoplasmic accumulation of α-syn after 30 days of differentiation, which could be quantitatively analyzed from anti-α-syn immunofluorescence intensity ( Figure 5C,D). We chose immunolabeling analysis with antibodies against α-syn because this technique has become the standard and most sensitive immunohistochemical method for neuropathological diagnosis of PD. 57 Treatment of parallel cultures of LRRK2-PD iPSC-derived DAns with 1 μM III for the last 48 h of differentiation resulted in a statistically significant decrease in overall α-syn immunoreactivity when compared with untreated cultures (Figure 5C−E).
Dendrimer effects were also evident when we quantified the percentage of DAns that stained positive for α-syn. Under our differentiation and culture conditions, DAns from LRKK2-PD patients accumulate α-syn in their cytoplasm (and are thus scored as α-syn-positive DAns) at approximately twice the frequency when compared with DAns from control individuals ( Figure 6A). Using DAns differentiated from two independent LRKK2-PD patients, we found that treatment with 1 μM III for 48 h was sufficient to reduce the percentage of α-synpositive DAns to values not statistically different from those measured in control DAns ( Figure 6A).
We next analyzed the capacity of dendrimers to reduce the intracellular accumulation of α-syn aggregates. Imaging by confocal microscopy of anti-α-syn immunostained DAns identifies α-syn aggregates as distinct puncta accumulated in the cell body. 58 In our experiments, the percentage of LRKK2-PD DAns containing α-syn puncta in the cell body was significantly higher than that of control DAns (51.8 ± 8.7% vs. 19.4 ± 4.7%, n = 5 and 4 independent differentiations using two iPSC lines per condition; Figure 6B−E). These results are in line with previous evidence 43,52,53 and highlight the advantage of using patient-specific iPSC-based models compared to other experimental systems where additional manipulations, such as treatment with exogenous α-syn fibrils or rotenone-induced α-syn fibrillation, are required. 22,40 Importantly, incubation of LRKK2-PD DAns with 1 μM III for 48 h reduced the percentage of cells displaying α-syn puncta to values similar to those shown by control DAns (Figure 6B−E). These results suggest that cationic carbosilane dendrimers of bow-tie topology (III), when used at non-toxic concentrations, are internalized by PD patients' DAns and directly interact with α-syn aggregates, probably helping to solubilize them into smaller aggregates that would be more easily degraded.

■ CONCLUSIONS
In summary, in this work, we demonstrate the effectiveness of cationic carbosilane dendrimers toward key PD-related cell phenotypes using an experimental system based on diseaserelevant cell types from PD patients. We further show the influence of dendritic topology on the interaction with α-syn and, therefore, on their ability to prevent the formation of αsyn aggregates. Bow-tie dendrimers showed good performance and were well tolerated by DAns, a cell type especially susceptible to toxic insults. 59 The development of new therapeutic approaches for PD requires the use of appropriate experimental conditions mimicking the neurodegeneration that takes place in patients. In this sense, iPSC technology 60 has opened unprecedented opportunities for the generation of genuinely human models of disease. 48 Dendrimers have been shown to interfere with the formation of amyloid fibrillary structures typically related with the onset and progression of protein misfolding diseases such as Alzheimer's disease 28,29,54 and prion diseases. 61,62 Thus, dendrimers as anti-neurodegenerative agents may have application in PD therapy in which disease progression is closely correlated to brain accumulation of insoluble α-syn. Numerous previous studies have explored the usefulness of dendritic systems to prevent and/or rescue α-syn aggregation in a variety of cell-free and cell-based experimental models of PD. 21,22,24,26,27 Our results lend additional support to this line of study and, importantly, validate the development of dendrimer-based anti-PD therapies using DAns, the cell type most relevant for the disease, obtained from PD patients. At the cellular level, our results confirmed a significant decrease in cytoplasmic levels of α-syn and in α-syn aggregates in DAns from LRRK2-PD-iPSC treated during 48 h with non-cytotoxic concentrations of bowtie dendrimer III. We believe that the results from our studies